Figure 3.

Microparticles released by Lm-infected macrophages stimulated DC maturation and resultant T cell proliferation. (a, b) Lm components were present in MPs. Macrophages were infected with CFSE-labeled Lm and the released MPs were isolated from the supernatants by centrifugation. The isolated MPs were analyzed by flow cytometry (a). Or, macrophages were labeled with PKH26 and infected with CFSE-labeled Lm. The green Lm components and red MPs were observed under two-photon laser scanning fluorescence microscopy (b). (c) Lm component-containing MPs stimulated DC maturation. DCs were incubated with PBS, MPs from Lm-infected or control macrophages for 24 h and were stained with CD80, CD86 or MHC class II mAb, the mean fluorescence intensity (MFI) was measured. *P<0.05, compared with PBS group. (d) DCs elicited T-cell activation after incubating with Lm component-containing MPs. Macrophages (5×10⁶) were treated with Lm or LPS and the released MPs were isolated from the supernatants. DCs were incubated with MPs and cocultured with T cells isolated from the spleens of Lm-infected BALB/c mice. The T-cell proliferation was measured. (e) Macrophages were treated with different MOIs, and the isolated MPs were used to treat DCs for T-cell proliferation assay as above. (f) The activation of MAPK and NF-κB of DCs by MPs. Bone marrow-derived DCs were stimulated with MPs from Lm-infected or control macrophages for various time intervals (0–60 min). Western blot was performed for analysis of MAPK ERK and IκB phosphorylation. Results are representative of at least three independent experiments. CFSE, carboxyfluorescein succinimidyl ester; DC, dendritic cell; ERK, extracellular signal-regulated protein kinase; IκB, inhibitor of kappaB; Lm, Listeria monocytogenes; LPS, lipopolysaccharide; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase; MOI, multiple of infection; MP, microparticle; NF-κB, nuclear factor kappaB; PBS, phosphate-buffered saline.